Gene Knockout Kits

Human and Mouse Knockouts Have Never Been Easier with XDel Technology.

EditCo’s Gene Knockout Kit was designed to increase the efficacy of CRISPR-mediated knockouts. To address the inefficiencies of traditional CRISPR knockout strategies, we developed a solution: a set of predesigned guide RNAs (gRNAs) targeting a single early exon of a gene that work cooperatively to increase editing efficiency by creating targeted fragment deletions.

Unlike the standard approach, which relies on a single gRNA to introduce random indels at one site—or multiple gRNAs that randomly target different regions—our method is specifically designed to create coordinated deletions that reliably disrupt the gene. This approach improves the likelihood of generating a complete functional knockout, reducing the unpredictability and inefficiency associated with traditional strategies.

In addition, the resulting knockout gene fragment can then be rapidly and accurately quantified using a standard PCR amplification to ensure seamless integration into your CRISPR workflow. Analyze your CRISPR edits in seconds using Sanger sequencing with our ICE tool.

Figure 1. XDel design includes up to 3 modified sgRNAs (grey bars) that target a single gene of interest. When co-transfected, the sgRNAs create concurrent double-stranded breaks at the targeted genomic locus and consequently induce one or more 21+ bp fragment deletions.

Figure 2. XDel multiple gRNA creates fragment deletions. Individual vs. XDel gRNA editing was compared for 2 gene targets (TNF, TLR4) in dendritic cells (transfected via nucleofection). Editing efficiency was analyzed by sequencing the targeted loci on a MiSeq and sequencing outcomes were categorized based on editing type (no indel, large deletion ≥50bp, small deletion <50bp, insertion). Data generated by Dr. Marco Jost and Dr. Jonathan Weissman, University of California, San Francisco, and Dr. Amy Jacobson and Dr. Michael Fischbach, Stanford University.

XDel Achieves Higher and More Consistent On-Target Editing in Immortalized, iPSC, and Primary Cells Compared to Single-Guide

Figure 3. XDel multiple gRNA on-target editing efficiency is significantly higher and more consistent compared to single-guide RNA methods. On-target editing efficiency of multi-guide (pink bars) vs their 3 respective single-guide RNAs (blue bars) across 7 endogenous target loci (with known high off-target editing sites) transfected in two immortalized (IMM) and two iPSC cell lines indicates that XDel technology delivers significantly higher and more consistent on-target editing efficiency compared to single-guide. Each transfection was performed in triplicate and data were analyzed via NGS by EditCo’s proprietary software analysis.

XDel technology maximizes on-target gene editing while minimizing off-target effects compared to single-guide editing

Figure 4. XDel multiple guide RNA off-target editing efficiency is significantly lower compared to single guide RNA methods when targeting endogenous sites known for their high off-target levels. Off-target editing efficiency of XDel cells (pink bars) at all 3 of their respective single-guide off-target sites vs their 3 respective single-guide RNAs (blue bars) across 63 off-target loci (3 off-target loci per 7 on-target sites tested) transfected in two immortalized (IMM) and two iPSC cell lines indicates that XDel cells deliver significantly lower off-target editing efficiency compared to single-guide. Each transfection was performed in triplicate and data were analyzed via NGS by EditCo's proprietary software analysis.

Figure 5. XDel multiple guide RNA editing efficiency remains high as gRNA concentration is decreased compared to single guide RNA methods. Average on-target (pink bars, multi-guide; blue bars, single-guide) editing efficiency in transfected HEK293 cells with multi-guide (left) vs single-guide (right) and SpCas9 at increasing RNP concentrations (0.25X, 1X, 4X) at 7 endogenous sites vs off-target editing efficiency (stacked grey bars) at 63 off-target sites indicates that XDel cells maintain high on-target editing efficiency with minimal off-target effects compared to single-guide. Each transfection was performed in triplicate and data were analyzed via NGS by EditCo's proprietary software analysis.

Edited Cells with XDel Technology Demonstrate Sustained Gene Knockout Across Multiple Cell Passages

Figure 6. High editing efficiency is sustained through multiple cell passages. Using EditCo’s multi-guide designs, up to 3 modified gRNA per target (across 6 genes) were conjugated with SpCas9, and RNPs were transfected into U2OS cells. Editing efficiencies were measured at different time points post-transfection and up to 4 passages. The median editing efficiency is >85 percent for each of the targets.

Cell Viability Is Maintained Across Passages with XDel

Figure 7. Cell viability is maintained through multiple cell passages in U2OS cells. Viability was taken using Celigo. Please note that CDK9 is a common essential gene, hence the lower viability and cell number observed along the passages.

XDel Technology Result in Sustained Protein Depletion

Figure 8. Protein depletion remains consistent across a variety of gene knockout cell lines. This enables direct and reliable use of these cell pools for functional assays. Western blot analyses for all 5 knockout target genes indicate a complete depletion of proteins across the 3 specified time points (days 7, 14, and 21) relative to the negative controls. Knockout cell pools for the target genes were generated by nucleofecting U2OS cells with multi-guide sgRNA and Cas9 (as RNPs). A negative control pool was also transfected for each target using non-targeting sgRNA.